Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
J Am Chem Soc. 2010 Mar 10;132(9):2952-60. doi: 10.1021/ja907706a.
An NMR approach is described which yields the methyl resonance assignments of alanine, threonine, valine, leucine, and isoleucine residues in proteins with high sensitivity and excellent resolution. The method relies on protein samples produced by bacterial expression using [(1)H,(13)C]-D-glucose and approximately 100% D(2)O, which is cost-effective and ensures the isotopic enrichment of all possible methyl groups. Magnetization transfer throughout the methyl-containing side chains is possible with this labeling scheme due to the high level of deuteration along the amino acid side chain, coupled with the selection of the favorable CHD(2) methyl isotopomer for detection. In an application to the 34 kDa periplasmic binding protein FepB 164 out of 195 methyl groups (85%) were assigned sequence-specifically and stereospecifically. This percentage increases to 91% when taking into account that not all backbone assignments are available for this system. The remaining unassigned methyl groups belong to six leucine residues, caused by low cross-peak intensities, and four alanine residues due to degeneracy of the (13)C(alpha)/(13)C(beta) frequencies. Our results demonstrate that NMR spectroscopic investigations of protein structure, dynamics, and interactions can be extended to include all methyl-containing amino acids also for larger proteins.
本文描述了一种 NMR 方法，该方法能够高灵敏度和高分辨率地确定蛋白质中天冬氨酸、苏氨酸、缬氨酸、亮氨酸和异亮氨酸残基的甲基共振分配。该方法依赖于使用 [(1)H,(13)C]-D-葡萄糖和大约 100%D(2)O 生产的细菌表达的蛋白质样品，这种方法具有成本效益，可确保所有可能的甲基基团得到同位素富集。由于氨基酸侧链上的氘化水平很高，并且选择了有利于 CHD(2)甲基同位素质谱检测的甲基异构体，因此这种标记方案可以实现含有甲基的侧链之间的磁化转移。在对 34 kDa 周质结合蛋白 FepB 的应用中，195 个甲基基团中有 164 个（85%）被特异性和立体特异性地分配了序列。当考虑到该系统并非所有骨架分配都可用时，这一比例增加到 91%。其余未分配的甲基基团属于六个亮氨酸残基，这是由于交叉峰强度低造成的，还有四个丙氨酸残基是由于 (13)C(alpha)/(13)C(beta) 频率的简并性造成的。我们的结果表明，蛋白质结构、动力学和相互作用的 NMR 光谱研究可以扩展到包括更大的蛋白质中的所有含甲基氨基酸。
